The ability to control expression of a cloned gene both temporally and quantitatively is critical to the study of its function. For two interacting genes or gene products, the ability to control both the timing and level of expression, independently, is essential. A research tool that performed this function would be novel, timely and of significant value to the research community. Toward this end, we have identified two ecdysone receptor (EcR) Ligand Binding Domain (LBD) mutants that bind two different small molecule agonist ligands, orthogonally. Each EcR(LBD) mutant has been fused to a different DNA Binding Domain (DBD) motif, GAL4 DBD and LexA DBD. Using these EcR fusions we have demonstrated their ability to control gene expression of two reporter genes in mammalian tissue culture, independently in a ligand- dependent manner. We propose here to further test the system in a variety of mammalian cell lines under different conditions. We also propose to engineer the components into a simple, two-plasmid format, with one plasmid containing all of the components that drive the system and the other plasmid acting as a cloning vector for two genes. Positive control reporter vectors and stable cell lines will also be developed to complement it. In Phase II we will develop the in vivo use of RheoPlex by determining the tissue distribution of the two ligands, activity of the receptors and construct transgenic mice. We will also use RheoPlex to explore the coordination of DNMT1 and G9a in methylating chromatin during DNA replication. 1 Precise temporal, spatial and quantitative control over the expression of transgenes via external application of small molecules is an extremely powerful tool for gene function analysis both in vitro and in vivo. In addition, it can greatly facilitate the production of therapeutic proteins and would be crucial for gene and cell therapy applications. With the recent availability of the genome sequences of a variety of mammals, scientific attention has transferred from gene identification to functional studies. The tool under development will provide cell and molecular biologists with a unique and valuable tool to study the function of more than one gene simultaneously within the same cell. This will greatly facilitate the in vitro and in vivo study of interacting genes, gene products, genetic pathways and biochemical pathways. [unreadable] [unreadable] [unreadable]